Abstract
The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na(+)/H(+) exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na(+)/H(+) antiport is discussed.
Highlights
The NADH:ubiquinone oxidoreductase, known as respiratory complex I,1 couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane [1,2,3]
Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids
We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration
Summary
The NADH:ubiquinone oxidoreductase, known as respiratory complex I,1 couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane [1,2,3]. In addition to the homologues of the 14 minimal subunits the mitochondrial complex I of eukaryotes contains at least 32 extra proteins but no additional redox groups [6]. Growth on glycerol and fumarate enabled growth of strain EP432 at about 450 mM NaCl [15] Under these conditions, the expression of the nuo genes coding for the subunits of complex I is 2-fold increased [18, 19]. It was shown that the overproduced subunit NuoL of the E. coli complex I mediates Naϩ uptake when reconstituted into proteoliposomes [20]. This transport was inhibited by the addition of EIPA, an inhibitor of Naϩ/Hϩ antiporters [21]. E. coli complex I is a primary proton pump but may be capable of secondary Hϩ/Naϩ antiport
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